Download Problem Set 3 (Due February 4th) 1. In 1896, Christiaan Eijkman

Problem Set 3
(Due February 4th)
1. In 1896, Christiaan Eijkman famously stated that “White rice can be poisonous!” Research this quote
and determine how it relates to the material covered in this unit. Be specific – which enzymes are
affected and how? Please note the date – this quote has nothing to do with the 2012 discovery that
some rice has elevated arsenic concentrations. Eijkman spent some time in Jakarta, Indonesia and
noted that many of the natives died from a disease we now know as beriberi. He made the connection
that this was likely due to the white rice heavy diet. White rice has the husk stripped from it to make it
easier to cook. By removing the husk, the nutritional content is also dramatically dampened (this is why
we now fortify rice and other crops). Of particular note, the natives had a notable Vitamin B1 (also
known as thiamin) deficiency. Thiamin is the precursor of the important cofactor TPP, which plays a
critical role in pyruvate dehydrogenase and -ketoglutarate dehydrogenase.
2. We know that pyruvate is ultimately oxidized to 3 equivalents of CO2.
a. Starting with the average oxidation number on each carbon in pyruvate, determine how many
electrons are produced during the oxidation to CO2. Does this make sense based on the
number of NADH and FADH2 that are produced?
b. Start with pyruvate. Number each carbon (C1, C2, C3). Trace the fate of each of these
carbons through a single TCA cycle.
c. How many cycles does it take for these three carbons to be completely removed from the
intermediate metabolites in the cycle (that is, at what point does C1 become CO2, at what point
does C2 become CO2, etc.). Please state any assumptions that you make about symmetry or
asymmetry in the intermediates/reactions.
3. Consider the conversion of Succinyl CoA to Succinate.
a. What enzyme catalyzes this reaction? Succinyl-CoA Synthase
b. Draw a mechanism for this reaction. Make sure to include the isolatable phosphohistidine
residue.
c. What provides the energy for GTP synthesis? Hydrolysis of the thioester
d. We often say that ATP is generated in this step. How can we make this statement when it is not
part of the overall chemical reaction? Substrate Level Phoshporylation – GTP readily
phosphorylates ADP
4. Oxaloacetate can by synthesized directly from pyruvate.
a. This is an example of an anapleurotic reaction. What does this mean? Replineshing – It
replenishes the intermediates of the TCA Cycle
b. Why is this not the optimal route for carbons from pyruvate to enter into the TCA cycle? This
route consumes an ATP and prevents the production of NADH; both are counterproductive to
energy production.
c. Accumulation of Acetyl-CoA activates this enzyme. Why does this make sense in the metabolic
scheme of the TCA cycle? If acetyl-CoA is accumulating, that means that citrate synthase is not
operating ideally. Perhaps this is because the [oxaloacetate] is below the Km, in which case
making more oxaloacetate would enable citrate synthase to function normally
d. Using the KEGG Pathway website, determine the name of the enzyme that catalyzes this
reaction, the gene code in humans, and any cofactors that are necessary in humans.
Enzyme: Pyruvate Carboxylase
Gene Code: 5091
Cofactors: biotin and Mn or Zn
e. Propose a mechanism for this reaction (I encourage you to be creative and come up with a
mechanism that makes sense based on your own chemical intuition – you will need to do this on
exams).
5. The standard free energies of hydrolysis for three high energy phosphate compounds are listed below.
Based on the structure or each molecule, justify the trend.
Phosphoenolpyruvate  pyruvate (G’º = -61.9 kJ mol-1), ATP  ATP (G’º = -30.5 kJ mol-1),
Phosphocreatine creatine (G’º = -43.1 kJ mol-1)
6. The typical intracellular concentrations of ATP, ADP, and Pi in muscles are 5.0, 0.5, 1.0 mM,
respectively. Using the values in problem 5, and assuming standard biological conditions:
a. What is G for the hydrolysis of ATP in these conditions?
b. Calculate the equilibrium concentration ratio of phosphocreatine to creatine in the creatine
kinase reaction
Creatine + ATP ⇌ Phosphocreatine + ADP
c. What concentration ratio of ATP to ADP would be required to yield equal concentrations of
phosphocreatine and creatine?
d. Assuming the concentration of Pi does not change, calculate the free energy of hydrolysis of
ATP under the conditions determined in 6c.
7. Recall that -Ketoglutarate is converted to Succinyl-CoA in a reaction very similar to pyruvate
decarboxylation. Please propose a mechanism for this reaction including important intermediate
structures and all cofactors that are involved.
8. Noting that each equivalent of NADH produced in glycolysis and TCA Cycle leads to the production of
2.5 ATP and each FADH2 produces 1.5 ATP (the topic of next week’s lecture), determine how many
equivalents of ATP are synthesized form one molecule of pyruvate.
9. Mitochondrial Isocitrate Dehydrogenase relies on allosteric regulation through a regulatory subunit to
activate or inhibit enzyme activity (the RCSB molecule of the month page does a good job at explaining
this). E. coli, which lack mitochondria, rely on a different mechanism to regulate enzyme activity.
Please read the attached paper and discuss how this E. coli enzyme is regulated and how this
mechanism relates to the ‘dietary’ carbon source. E. coli Isocitrate Dehydrogenase (IDH) can be
inhibited by the activity of another enzyme, IDH kinase/phosphorylase (gene aceK). This enzyme,
which is part of an operon containing genes for Malate Synthase and Isocitrate Lyase (the two enzymes
of the glyoxylate bypass), can inactivate IDH through phosphorylation. This inactivation of IDH
decreases flux through the Krebs Cycle and, in turn, increases flux through the Glyoxylate Bypass.
This bypass is particularly important because, when C2 units (e.g. acetate) are the primary carbon
source, the Krebs Cycle would result in an immediate conversion of both carbons to CO2; this alternate
pathway allows E. coli a way to ensure that some of the dietary carbon is reserved for anabolic
pathways (instead of being converted to CO2). This observation is reinforced by the fact that E. coli
strains that lack aceK are not able to grow on acetate.
Perhaps you spent some time investigating the Glyoxylate Pathway in your book. If so, you would note
that this cycle incorporates acetate in two reactions. The four carbon atoms exit the pathway as the C4
intermediate succinate, which can be readily converted to other Krebs Cycle intermediates and serve
as building blocks of other metabolites.